Production of bile acids

ABSTRACT

This invention relates to new and novel methods for the production of substantially pure bile acids, and particularly, 3 Alpha ,7 Alpha -dihydroxy 5 Beta -cholanic acid, from natural sources thereof.

United States Patent Saltzman Nov. 11, 1975 1 PRODUCTION OF BILE ACIDS3.836.550 9/1974 Jones et m. .l 260mm [75] ltlVEfltOl'i William H.Saltzman, NGW OTHER PUBLCATIONS Rochelle. NY.

[73] Ass|gnce1 Intellectual Property Development Hoffmmn f f 5cm pp 7Corporation, New Rochelle NY Journal of Biochemistry-Vol. 38 pp. 93-98U951). [22 Filed: N0 19 Journal of Bi0chemistry\*'ol. 70. pp 235-29l 21Appl. NO.;417,170

Related U.S. Application Data [63] Continuution-in-purt of Ser. No290.910. Sept II 19714 abandoned,

[52] US. Cl. t. 260/3971 lSll Int. Cl. C07J 9/00 {58] Field of Search260/397.l

[56] References Cited UNITED STATES PATENTS 2.745.849 5/1956 Fogle r l rt r 26U/397.l

Merck Index. 8 Ed. pp. Z29 & 230. N68.

Prinuu' E.\'uminerElbert L Roberts [57] ABSTRACT This invention relatesto new and novel methods for the production of substantially pure bileacids. and particularly. 3a,7a-dih vdroxy 5B-cholunic acid. from naturalsources thereof 18 Claims. No Drawings PRODUCTION OF BILE ACIDS ThisApplication is a continuation-in-part application of my previouslyfiled, copending application, Ser. No. 200,9"), filed Sept. 21, 1972,now abandoned.

This invention relates to and has as its object, the production ofsubstantially pure bile acids from natural sources thereof, and to newand useful processes therefore.

More particularly, this invention relates to new and novel methods forproducing substantially pure bile acids, and particularly3a,7B-dihydroxy-SB-cholanic acid, from natural animal sources thereof.Bile acids are naturally occurring steroidal compounds, which arenaturally synthesized by various animals. The most common of thesenatural bile acids are those possessing the following structure:

COR

wherein R is a carboxyl group and hydroxyl substitu ents may be found inthe 3, 7 and/or 12 positions, depending on the particular bile acidinvolved.

In nature, bile acids are synthesized in the liver of animals and areincorporated into the bile, which is a fluid secreted by the liver andpoured into the intestine of the animal. The bile acids are the majorconstituent of the bile and usually produced in various numbers andquantities by different animals, all of which are incorporated into thebile. Among the common animal originated bile acids may b included:SaJa-dihydroxy-SB- cholanic acid; 3a,7a,lZa-trihydroxy-5B-cholanic acid;and 301,12a-dihydroxy'5B-cholanic acid.

Due to the fact that the animal incorporates all these bile acids and anumber of other materials, for example, pigments, lipids and proteinmaterials, into the bile, it has heretofore, been inconvenient anduneconomical to isolate any particular bile acid from animal bile andtherefore, many individual bile acids, even though naturally occurringin some abundance, had tobe prepared synthetically fromvariousprecursors. In addition, heretofore, it has not been possible toproduce certain bile acids in a sufficiently pure from for various used,for example medical thereapy. V

l have now discovered a method whereby individual bite acids, insubstantially pure form, can be produced economically and efficiently,directly from natural animal bile. Most importantly, for purposes ofthis invention, I have discovered an economically efficient method forproducing substantially pure 3a,7a-dihydroxy-SB-cholanic acid fromanimal bile. The use of the term, substantially pure, in thisspecification and the claims appended thereto, is meant to denote achemical purity in. excess of 99.0%, as evidenced by standard andacceptable analytical procedures. Heretofore, it has not been possibleto produce substantially pure bile acids directly from natural animalbile.

The animals which may be employed as the source of natural animal bilein the practice of this invention, are

those whose bile is rich in the desired bile acid. Among those animalswhich may be included in this group are such animals as birds, forexample, chickens, turkeys, geese, pheasants, owls an dducks', mammals,for example, oxen, rats, guinea pigs, bears and pigs; and fish, forexample, cod fish, mullet fish, anchovies and harder fish. The mostpreferred source of the natural bile are animals, the major constituentof whose bile is the desired dihydroxy bile acid, and these include suchbirds as chickens, turkeys, geese and ducks.

The process of this invention involves a number of steps commencing witha suitable animal bile as starting material. The animal bile, forexample chicken or turkey bile. may be collected in any manner known toand convenient to the skilled worker, with the usual precautions beingtaken to avoid damage or deterioration of the animal bile startingmaterial.

The successful practice of this invention involves two major proceduresin the treatment of the natural animal bile to obtain the desiredsubstantially pure bile acid. The first of these major proceduresinvolves the removal from the natural animal bile of the undesiredimpurities and other extraneous materials, such as pigments, lipids andprotein substances found therein. The second major procedure involvesthe isolation and purification of the desired bile acid, after all theundesired impurities have been removed.

REMOVAL OF IMPURITIES FROM NATURAL ANIMAL BILE The removal of theundesirable impurities from natural animal bile including bile pigments,lipid impurities and the like, can be achieved by a number of new andnovel procedures. One such method involves the direct solvent extractionof these impurities with an organic solvent capable of solubilizing thebile acids in the bile, and in which solvent the impurities areinsoluble, for example, an alcohol, such as methanol or ethanol; aketone, such as acetone; or an acid, such as, acetic acid; and otherlike solvents. The undesired impurities, due to their insolubility inthe solvent, are caused to precipitate out of the solution and may thenbe removed by simple filtration. Although this method has given somewhatsatisfactory results, it has been found that its practice does notremove substantially all the impurities and further purification isrequired to obtain removal of substantially all of the undesiredmaterial from the bile. Another Substantially to remove the unwantedimpurities requires the natural bile to first be mildly hydrolyzed as bytreatment with a base, such as an alkali metal base, for example, sodiumhydroxide or potassium hydroxide. The hydrolyzed material is thenneutralized and extracted with a suitable solvent system, for example,chloroform: methanol, in which the bile acids portion of the bile issoluble in one solvent, but the impurities are not, thus causing theimpurities to be separated out into one solvent of the system and thus,rendering them subject to withdrawal therewith. This method, althoughmore satisfactory than that set forth hereinabove, is also not capableof the removal of all of the impurities from the natural bile andadditional purification is required to obtain the optimum final productsof this invention.

Still another method that has been found to remove the impurities fromthe natural bile may be employed in the practice of this invention. Thismethod involves first, mildly hydrolyzing the crude natural bile with abase. such as an alkali metal base, for example, NaOH or KOH,neutralizing the resultant hydrolyzed solution, and then dehydratingsaid solution, as by spray drying, lyophilization, drum drying, membraneseparation or freeze drying. Although the hydrolysis procedure may behelpful in the removal of certain of the impurities, in certain cases,for example in chicken or turkey bile, l have found that the hydrolysisprocedure can be dispensed with, and the natural animal bile isdehydrated as the first step in the process. By omitting the hydrolysisprocedure in these instances, I have found that the quality andcharacter of the final product is not adversely effected.

In the most preferred process of this invention, the natural crudeanimal bile is first dehydrated by any of the well known methods, suchas those enumerated above. It is preferred that the dehydrationprocedure be accomplished by freeze drying, or lyophilization, althoughthe other well known methods also yield equiv alently satisfactoryresults.

The resultant dehydrated natural bile material is then esterified as bytreatment with a suitable esterification agent. Depending upon theanimal which is the source of the natural bile, the esterification agentemployable in the practice of this invention, may be altered.Preferably, the esterification agent employed in this invention is analcohol, which may be a hydrocarbon alacohol or may be a substitutedalcohol. Among the alcohols which may be employed as esterificationagents herein may be included such saturated or unsaturated alcohols asthe alkanols, for example, methanol, propanol or butanol; the alkenols,for example, ethenol, or propenol; the cyclic alcohols, for example,cyclohexanol, or cyclooctanol; the aryl alcohols, for example, phenol,benzyl alcohol, cinnamyl alcohol, and other like alcohol esterificationagents. In addition, the alcohols of this invention may be substitutedor unsubstituted and may contain such substituents as halides, forexample, chlorides or bromides, or sulfur, or oxygen. without departingfrom the practice of this invention. Most preferably, l have found thatesterification of the natural bile material may be satisfactorilyaccomplished by treatment thereof with an alkyl esterification agent,such as methyl esterification agent, for example, methanol in an acidmedium, such as a mineral acid, for instance, sulfuric acid. Where analkyl esteriflcation agent is employable in the practice of thisinvention, other alkylating agents may also be employed. Thus, suchalkylating agents as diazomethane, may also be employed in the practiceof this invention.

The esterification of the natural bile material yields the esters, forexample the alkyl esters, of the bile material impurities, such aspigments, etc., whereupon they are apparently rendered soluble incertain organic solvents and insoluble in others. Likewise, the bileacids are present in the bile are soluble in certain solvents and not inothers. By subjecting the resultant esterified composition to the propersolvent system, it has been found to be possible to separate the bileacids from the impurities. Among the satisfactory solvent systems whichmay be employed in the practice of this invention are the halogenatedhydrocarbon organic solvents such as chloroform, methylene chloride,methylene dichloride, such aromatic organic solvents such as, benzene,toluene, and the like, and such organic solvents as alcohols, forexample, methanol and ethanol, alkanes, such as hexane or heptane,ketones, for examplc. acetone. benzophcnone. or cyclohexanone, ethers,such as methyl or ethyl ether, carboxylic acids, for example. aceticacid, and other similar suitable organic solvents. In practice ofthisinvention, it has been found that a satisfactory solvent system is onewhich is comprised of at least two phases, in one phase of which thebile impurities are soluble and the remaining phases in which the saidimpurities are insoluble. Conversely, the desired bile acid fraction ofthe bile must be insoluble in the phase in which the impurities aresoluble, while being soluble in the remaining phase. I have now foundthat a solvent system comprised of the following phases may besatisfactorily employed in the practice of this invention: halogenatedhydrocarbonzalcohol, for example, chloroformzmethanol,chloroformzethanol, methylene chloridezmcthanol; aromatic organicsolvent:alcohol, for example, tolucnezmethanol, benzenezmethanol;aromatic organic solvent:alkane, for example, benzene:hexane,toluenezhexane; etherzalcohol, for example, methyl etherrmcthanol;halogenated hydrocarbonzketone, for example, chloroform-acetone andother like organic solvent systems which the skilled worker maydetermine to be employable in the practice of the instant invention inconformance with the teachings set forth herein.

It has been found that the most economically satisfactory results may beobtained in the practice of this invention when the solvent systememployed is comprised of a halogenated hydrocarbon phase and an al coholphase. More particularly, a solvent system comprised of a halogenatedhydrocarbon, for example, chloroform, and an alcohol, for examplemethanol, has provided very satisfactory results in the practice of thisinvention. The bile acid fraction of the crude bile is not soluble inthe halogenated hydrocarbon solvent phase, while it has beenunexpectedly been found that substantially all the esterified impuritiesare. It has also been found that for the most successful practice ofthis invention, it is desirable to process the dehydrated bile materialunder substantially anhydrous conditions. It appears that the presenceof material amounts of water, either in the bile material or in thesubsequent processing thereof results in a product of relativelyunsatisfactory quality.

The vigorous admixing of the solvents and the bile, and the addition ofaseparating phase to the system, if desired, results in completelyseparated phases, one containing the undesired impurities and the othercontaining the bile acid fraction. Among those materials which may beemployed as a separating phase, if one is desired, may be includeddilute mineral acids, for example, hydrochloric or sulfuric acids, orwater. Thus, all the impurities which are found in one solvent phase maybe drawn off therewith, leaving only the bile acid solution, free ofimpurities, to further purify hereunder. This method of removal of theundesired impurities from the natural bile is the most preferred in thepractice of this invention, and has been found to result in the mostsatisfactory final products hereunder. ln practice, I have found thatwhile the impurity containing solvent phase has a deep greenish, orgreenish-black color, the bile acid containing solvent phase is almostwater-white, and contains practically no detectable amounts of undesiredimpurities, such as pigments.

In nature, natural animal bile exists in a peptide conjugated form, i.e.the natural acids have, by the animal system, been conjugated with aminoacids. The two principle amino acids with which the natural animal bileacids have been conjugated are glycine and taurine. In the case of theglycine conjugated bile acids, it has been found that a slight variationin the process by which the impurities and pigments are removed from thenatural bile, as set forth hereinabove, can also provide satisfactoryresults. I have found an additional procedure whereby substantially allof the pigments and other undesired impurities in glycine conjugatedbilc can be removed, all without departing from the ambit of the instantinvention.

The natural bile is brought to substantial dryness and esterified asaforesaid. However, the esterification is permitted to continue untiland to the extent that the pigment fraction, the other impurity fractionand the bile acid fraction of the natural bile are all obtained in theiresterified form. The esterification agents enumerated hereinabove mayalso be employed in the instant procedure. The thus completelyesterified natural bile is then subjected to a selective hydrolysisreaction, wherein only the esterified bile acid fraction of the naturalbile has been hydrolyzed. This selective hydrolysis may be accomplishedby subjecting the estcrifiedbile to the commonly employed hydrolysisprocedures in the presence of a suitable pH adjusting buffer. It hasbeen found that this selective hydrolysis procedure may be effectivelycarried out where the hydrolysis reaction mixtures pH is kept eitherhighly basic, or highly acidic, for example, a pH of about 9-12, orabout l3, respectively, the conditions of hydrolysis being ad justedaccordingly, as is well known to the skilled worker. The selectivehydrolysis reaction is continued until the esterified bile acid fractionof the bile is hydrolyzed to the free acids, and the resultanthydrolyzed bile is then subjected to the solvent separation procedure asset forth hereinabove, to yield the desired solvent phase containing thesubstantially pigment and impurity-free bile acid fraction of thenatural bile. The solvent phase containing the bile acids may then befurther treated by the processes of this invention to yield the desiredfinal products thereof. The bile acid containing solution may thus beneutralized, and the solvent evaporated off to yield a crude bile acidcomposition which may then be further treated hereunder.

ISOLATION AND PURIFICATION OF THE DESIRED BILE ACID The crude bile acidcomposition thus obtained, may then be treated to obtain the desiredfinal pure bile acid of this invention. One method that may be employedinvolves the hydrolysis of the crude bile acid composition to split offthe amino acid conjugates, if any, in which form the naturally occurringbile acids are found. This hydrolysis may be accomplished by treatingthe bile acid composition with a hydrolyzing agent, for example analkali metal base, such as KOH or NaOH. The resultant hydrolyzedmaterial may then be acidified as by treatment with a mineral acid,suchas I-ICI or H 50 the acidified solution extracted with a suitableorganic solvent, such as ethyl acetate, ethyl ether, isopropyl ether, ordiisopropyl ether, and the resultant solution is reduced to dryness toyield a crude residue of the free bile cids.

This crude residue may then be esterified by treat ment with a suitableesterification agent. Preferably, the esterification agent employed isone which will yield an alkyl esterified product, and may be an alcoholwhich is a hydrocarbon alcohol, which may be substituted orunsubstituted. Among the alcohols which have been found to providesatisfactory results hereunder. may be included such saturated orunsaturated alcohols as the alkanols, for example, methanol, propanol orbutanol', the alkenols, for example, propenol; the cycloalkanols, forexample, cyclohexanol; the aryl alcohols, for example, phenol, benzylalcohol, cinnamyl alcohol, and other like alcohols. In addition, thealcohols employed herein may be substituted or unsubstituted andthereof, may contain such molecular substituents as halogens, forexample, chlorine or bromine, or sulfur, without altering thesatisfactory results obtained hereunder. In addition, furtheresterification agents may be employed in the practice of the foregoingpro' cedure, particularly in the case where the objective thereof is thealkyl esterification of the substrate. Thus, such alkyl esterificationagents such as diazomethane, and other like known agents may also besuccessfully employed in the practice of this invention.

Most preferably, in the practice of this invention, the crude residueobtained above, is subjected to alkyl esterification although the otheresters thereof may also be employed herein.

This crude residue is then alkyl esterified as by treatment with analkyl esterification agent, such as methanol in an acidic medium, forexample, sulfuric acid, or diazomethane in ether, to yield the alkylesters of the bile acids. These alkyl esters may then be subjected tocolumnar extraction, for example in a column of alumina, employing asuitable solvent system, such as ethyl acetate-benzene, to yield thecrude alkyl ester of the desired bile acid, i.e.,3a,7a-dihydroxy-SB-cholanic acid. On further elution with a suitablesolvent system, for example, methanol-ethyl acetate, additional crudealkyl ester of the 3a,7adihydroxy-5B-cholanic acid, combined in the caseof the bile source being chicken or turkey bile, with the alkyl ester of3a,7a,l2atrihydroxy-SB-cholanic acid. The second eluate may then berecycled through the extraction column and with the proper solventsystem, for example, ethyl acetate-benzene, yield further amounts of thecrude alkyl ester of 3a,?a-dihydroxy'5B-cholanic acid.

The resultant crude alkyl ester of the desired bile acid is thenhydrolyzed, as by treatment with a suitable base, for example an alkalimetal base, such as, KOH or NaOH, and the resultant product acidified,extracted with a suitable solvent, filtered and dried. The resultantresidue is then treated with a suitable solvent, such as ethyl acetate,at elevated temperature, and the resultant solution, upon cooling, isfound to contain substantially pure crystallised 3a,7a-dil.;,u'roxy-SB-cholanic acid, which can be recovered by filtration.

Still another method has been found for obtaining substantially purebile acids hereunder. After hydrolysis of the impurity free residueobtained as set forth hereinabove, to obtain the crude residue of freebile acids, this crude bile acid residue can be processed by a methodwhereby there is obtained a selective insoluble salt precipitation ofthe bile acid to yield the desired bile acid directly.

After removal of the impurities and the hydrolysis of the peptideconjugated bile acids, the resultant solution containing the free bileacids is treated with a suitable salt, which will cause a selective,differential bile salt precipitation in a suitable solvent. In otherwords, a suitable salt must be employed which will cause the formationof free bile acid salts which will possess differential solubilities ina selected solvent. Thus, where the free bile acids are3a,7a-dihydroxy-SB-cholanic acid and 311,701,]Za-trihydroxy-SB-cholanicacid, as in chicken, turkey and goose biles, the suitable salt which maybe employed is one which will cause the resultant bile acid salts tohave differential solubility properties in a particular solvent. Thesalts which have now been found to be employable in the practice of thisinvention include such salts as Group Ila metal salts. The Group llametal salts which may be employed in the practice of this inventioninclude such salts as barium, strontium, and calcium salts, for example,Group lla halide salts, such as the chloride salts, such as bariumchloride, or Group II a acetate salts, or Group Ila nitrate salts. Thefree bile acid composition is treated with the suitable Group lla salt,for example barium chloride, first in an aqueous solution to form thebile acid salts and then in a suitable solvent in which one of theresultant bile salts is soluble, while the other is not. in the case ofthe bile acids derived from chicken bile or turkey bile hereunder. abarium chloride salt and an organic solvent, for example, an alcohol,such as methanol, may be employed. The desired bile acid salt is thusseparated, and may then be treated with an acid, such as a mineral acid,for example, HCl, to dissociate the salt and obtain the desiredsubstantially pure free bile acid product. The resultant substantiallypure bile acid final product may then be further purified if desired, asby recrystallization from a suitable solvent, and then dried to yieldthe substantially pure, free bile acid final product of this invention.

In addition to the foregoing, various other suitable organic solvents inwhich the resultant Group lla bile acid salts will have a differentialsolubility, thus enabling their separation, may be employed in thepractice of this invention. The skilled worker can easily determinethose organic solvents which may thus be employed in the practice ofthis invention, which include such organic solvents, as ethyl acetate,hexane, and acetic acid, among others, all of which may besatisfactorily employed herein.

Further to the forgoing, the concurrently produced bile acid salt, whichis not separated and purified hereunder. which in the case of chickenbile is 301,701,120:- trihydroxy-SB-cholanic acid, may also be furtherpurified and treated in accordance with well known procedures to yieldits respective free bile acid. Once isolated in its free bile acid form,in the case ofchicken bile, the 302,701,l2a-trihydroxy-SB-cholanic acidthus obtained, may be further treated as by the method described byFeiser et al., in Volume 72, Journal of the American Chemical Society,page 5530, (1950), to yield additional amounts of the desired3a,7a-dihydroxy-SB- cholanic acid.

Although the foregoing general description of this invention has beensomewhat limited to a discussion of the treatment thereunder of naturalbile containing material amounts of 3a,7a-dihydroxy-SB-cholanic acid, itshould be understood that the principles and practice thereof is notlimited to such natural bile and is generally applicable to all naturalbile. l have found that with merely minor modifications easilydeterminable by the skilled worker in view of the teachings anddisclosures hereof, the process for removal of the undesired impuritiescontained in natural bile is generally applicable to all natural animalbile and hence the process of this in- 3a7a-dihydroxy-SB-cholanicvention may also be employed to separate and purify the naturallyoccurring bile acids contained in such natural animal bile as may beobtained from oxen, pig, possum, sheep, and man. The practice of thisinvention with such natural bile starting materials, including theanimals listed hereinabove, and such laboratory animals as rats, guineapigs and the like, will result in equivalent satisfactory production ofthe requisite substantially pure bile acids of this invention.

l have found in the practice of this invention that the resultantsubstantially pure bile acid, and particularly, the3a,7adihydroxy-SB-cholanic acid produced hereunder, are the pureset bileacids 1 have seen. The purity which has been checked by thin layerchromatography, gas liquid chromatography and melting point, has beenshown to be in excess of 99+% and the melting point appears to be higherthan any found in the literature, thus indicating a final product ofextraordinary purity. Obviously. in those applications where thecompound may be employed for medical uses, purity of the product is ofutmost concern.

The invention is more particulary described and set forth in thefollowing Examples:

EXAMPLE I Ten ml. of chicken gallbladder bile are added to 200 ml. ofhot methanol with stirring. After cooling, the resultant precipitate wasremoved by filtration and the alcoholic solution evaporated to drynessat 60C., in vacuo. The resultant dry residue was then dissolved in 25ml. of 10% aqueous NaOH and the solution autoclaved at 14 lbs/sq.in. for3 hours. The solution is then cooled in an ice bath, acidified to pH 1.0with 4N HCl, and extracted 4 times with 50 ml. of ethylene acetate. Theorganic solvent fractions are washed with water to near neutrality anddried over Na SO The organic phase is then filtered off and evaporatedto dryness. The resultant material is then dissovled in 25 ml. ofanhydrous methanol containing 2% cone. H allowed to stand overnight andan equal volume of water is added. The resultant solution is thenextracted 3 times with 50 ml. diethyl ether-benzene (2:1v/v) and theorganic phases combined, washed with 5 ml. of water, 5 ml. of saturatedNaHCO and again 3 times with 5 ml. of water. The solution is dried overNa SO filtered and evaporated to dryness. The resultant residue is thendissolved in 50 ml. ethyl acetate-benzene (1:9v/v) and placed on acolumn of alumina, grade III, 200 grams. The column is washed with 400ml. ethyl acetate-benzene (3:7v/v) to remove colored impurities. Themethyl ester of 3a,7a-dihydroxySB-cholanic acid is then removed from thecolumn with 800 ml. ethyl acetate-benzene (4:6v/v), and the solventevaporated off in air at 60C. The resultant residue is then dissolved in50 ml. of 5% KOH in methanol and heated to 60C. for 1 hour. Theresultant solution is cooled in an ice bath and acidified to pH 1.0 with4N HCl. The resultant acidic solution is diluted with 100 ml. of waterand extracted 4 times with 75 ml. of ethyl ether, and the etheralsolution washed with water to pH 5.0, dried over Na,SO filtered andevaporated to dryness. The resultant residue is then dissolved in 30 ml.of hot ethyl acetate and allowed to stand at room temperature. Uponcooling, crystallization occurs, and after filtration on a Buchnerfilter, with suction. and drying overnight in vacuo, there is obtained447 mg. of acid, melting at 9 l42-l44C., analyzed as pure material bythin layer chromatography using the upper phase of a solvent system,toluene-acetic acidwater, (:5:1 v/v/v) and 50% H 50. in water asdetecting agent, The yield was calculated at 54.7%.

EXAMPLE ll 50 ml. of crude chicken gallbladder bile was lyophilized todryness to yield a dry greenish-black powder. This material was thendissolved in 100 ml. of anhydrous methanol and the solution heated tonear boiling. The hot solution was then filtered and the greenprecipitate washed with ml. of hot anhydrous methanol and discarded. Themethanol wash was combined with the original methanol solution, 2 ml. ofcone. H 80 was added and the solution allowed to stand overnight at roomtemperature. The solution was then transferred to a separatory funnelcontaining 200 ml. of chloroform and ml. of water, and the funnel shakenfor 1 minute to permit separation of the phases. The lower chloroformlayer was a dark greenish color and contained all the pigment and otherimpurities, while the upper alcoholic layer was water-white andcontained the bile acid fraction. The chloroform layer was drawn off anddiscarded.

EXAMPLE Ill 50 ml. of crude chicken bile was lyophilized to dryness toyield a dry greenish-black powder. This material was then dissolved in100 ml. of methanol and the solution heated to near boiling, and thenfiltered. The resultant greenish-black precipitate was discarded. Thefiltered methanol solution was then treated with 5 gm. of KOH added atroom temperature, with stirring, and the resultant solution allowed tostand overnight and was then neutralized with cone. H,SO with stirring.After neutrality was reached, 2 ml. of cone. H 80 was added andthesolution allowed to stand overnight at room temperature. The solutionwas then cooled in an ice bath with 20 ml. of ice water and saturatedsolution of sodium bicarbonate was added dropwise until a pH of 8.0-9.0was reached. The solution was then filtered and transferred to aseparatory funnel containing 200 ml. of chloroform and 20 ml of waterand shaken for 2 minutes to permit separation of the resultant phases.The lower chloroform layer was greenish in color and contained theundesired pigment and lipid impurities, while the upper methanol layerwas near water white and contained the bile acids.

EXAMPLE IV Fifty ml. of native chicken gallbladder bile is stirred with2 gm. of NaOH until dissolved and allowed to stand overnight. Theresultant solution was then cooled in an ice bath and 4N HCl was addeddropwise with stirring until the pH was below 2.0. An additional 1 ml.of conc. HCl was then added. To the resultant solution with stirring,was added dimethoxypropane at the rate of9 ml per 1 ml. of solution, atroom temperature, and stirring was continued for one hour and theresultant solution allowed to stand overnight. Thereafter, the solutionwas cooled in an ice bath, 10 ml. of ice water was added, and then asaturated aqueous sodium bicarbonate solution was added until the pHbecame alkaline. The resultant solution was filtered and transferred toa separatory funnel containing 200 ml. of chloroform and shaken for oneminute, after which the phases were allowed to separate. The lowerchloroform phase contained the unwanted impurities and was drawn off anddiscarded. The upper, colorless phase contained the bile acids inaqueous methanol-acetone.

EXAMPLE V The clear solution obtained in Example ll above, wasconcentrated by distillation to remove most of the organic solvents. Theresidual aqueous solution containing peptide conjugated bile acids wasdiluted with water containing sufficient NaOH to result in 100 ml.ofsolution containing 10% NaOH. The resultant colorless alkalinesolution was then autoclaved at 14 lb/sq. in. for 3 hours, and theresultant hydrolyzed solution colled in an ice bath and acidified with4N HCl, dropwise with stirring, to yield a light yellow gummyprecipitate. The precipitate was filtered off, washed with water andthen dissolved in ml. of 10% aqueous ammonium hydroxide with stirring,and then heated to near boiling. To the stirring solution was addeddropwise, 40 ml. of a 10% barium chloride solution, yielding a whitecrystalline precipitate. The precipitate was filtered off. washed withmethanol and dried in vacuo at 60C., yielding a crude barium salt of3a,7a-dihydroxy S-B-cholanic acid, weighing 4.9 gm., which was thensuspended in ml. of ethyl acetate, 100 ml. of 3N HCl was then added andthe three phase system shake until the precipitate was dissolved. Theethyl acetate layer was separated, the aqueous acidic layer was washedwith 50 ml. of ethyl acetate and the ethyl acetate aliquots combined.The combined ethyl acetate solution was washed to neutrality with waterand dried over sodium sulfate, and the aqueous layers were discarded.

The ethyl acetate of the resultant solution was then evaporated off andthe residue was dissolved in 100 ml. of methanol and 10 ml of 10% bariumchloride solution was added with stirring followed by 1 ml. of cone.ammonium hydroxide, added dropwise with stirring. The resultant solutionwas brought to boiling, and allowed to cool to room temperature andfiltered.

The filtrate was tested for completeness of precipitation by theaddition of an amount of barium chloride solution and any precipitateformed was treated as above, to yield additional precipitate which wasthen added to the original precipitate and the combined material wasthen washed with methanol and dried in vacuo at 60C., yielding a crudebarium salt of 3a,7a-dihydroxy-5B-cholanic acid, weighing 4.1 gm.

The crude barium salt thus obtained, was then shaken with 100 ml of 3NHCl and 100 ml of ethyl acetate until complete dissolution was obtained.The phases were allowed to separate, the ethyl acetate phase was drawnoff, the aqueous acidic phase washed with ethyl acetate and the ethylacetate aliquots combined, washed to neutrality with water and driedover sodium sulfate. The aqueous layers were discarded.

The washed and dried ethyl acetate solution of3a,7a-dihydroxy-5B-cholanic acid thus obtained, was then evaporated to avolume of 40 ml. at 50C. under a stream of air. The resultant solutionwas then cooled to 4C. and n-hexane at 4C. was added until the solutionbecame slightly turbid. The ethyl acetate-n-hexane solution was thenallowed to stand overnight at 4C.. causing pure3a,?a-dihydroxy-SB-cholanic acid to crystallize on the walls of theflask. The crystals were collected by filtration and washed withn-hexane, and

dried in vacuo at 60C. yielding 3.2 gm. of pure3a,7a-dihydroxy-SB-cholanic acid. (99.0% pure by gas liquidchromatography) having a melting point of l40-l42C.

Further purification of the product may be obtained by following theprocedures set forth in the succeeding examples VI to lX.

EXAMPLE VI The product obtained in example V is dissolved in ethylacetate to which n-hexane is added, and the solution is subjected tofive plate countercurrent distribution between 25 ml. of 70% acetic acid(aqueous) and 40 ml. of 405 isopropyl ether in n-hexane' v/v. Thepurified bile acid product will be in the isopropyl ether phase fromwhich it may be recovered by evaporation.

EXAMPLE VII The product obtained in example V above, may be furtherpurified by column partition chromatography on Celite columns containing70% acetic acid as the stationary phase, 40% isoproyl ether-n-hexane asthe mobile phase. A lgm. column purifies 1 gm. of the crude bile acid.

EXAMPLE VIII The product obtained in Example V about, may be furtherpurified by adsorption chromatography on acidic alumina, activity gradeV. The crude bile acid is dissolved in ethyl acetate or acetone and putthrough the column, which adsorbs the impurities and allows the purebile acid solution to pass through.

EXAMPLE IX The crude bile acid product obtained in example IV above, maybe esterified with either diazomethane or methanol in H 80 as describedin example 1 above, and the esterified material purified on an aluminacolumn as fully set forth in example 1, above.

EXAMPLE X The procedure of Example V was followed to the point where 4.9grams of dry crude barium salt of 3a,7a-dihydroxy-SB-cholanic acid isobtained. To this dry crude material was then added 100 ml. of absolutemethanol and the solution heated to near boiling with stirring, untilthe crude material completely dissolves. The heated solution was thencooled whereupon white crystals formed. The solution was filtered anddried to yield the barium salt of the 3a,7a-dihydroxy-5B- cholanic acid,weighing 4.0 gm. and more than 98% pure barium salt. The barium saltthus obtained was then shaken with 100 ml of 3N HCl and 100 ml. of ethylacetate until complete dissolution was obtained. The phases were allowedto separate, the ethyl acetate phase was drawn off, the aqueous acidicphase washed with ethyl acetate and the ethyl acetate aliquots combined,washed to neutrality with water and dried over sodium sulfate.

The washed and dried ethyl acetate solution was then evaporated to avolume of 45 ml. at 50C. under a stream of air and the resultantsolution cooled to about 4C. and held there overnight, causing pure30:,7or-dihydroxy-SB-cholanic acid to crystallize. The crystals werecollected and dried in vacuo to yield 3.1 gm. of pure3a,7a-dihydroxy-SB-cholanic acid (99.0+% pure by gas liquidchromatography), having a melting point of I4ll43C.

The foregoing procedure may also be successfully practiced withequivalent amounts of anhydrous ethanol, acetone or acetic acid beingsubstituted for the absolute methanol solvent.

EXAMPLE Xl The procedure of example V is followed, except thatequivalents amounts of n-heptane or n-octane or npentane are substitutedfor the n-hexane yielding like results.

EXAMPLE XII The procedure of example ll was follwed except that naturalanchovy, harder fish, codfish or sheep bile was substituted for thechicken gallbladder bile, yield a clear light colored solution which issubstantially free from impurities and contains substantially all thebile acids of the respective bile.

EXAMPLE XII] The method of examples I, II, III, IV and V are followedexcept that turkey gallbladder bile, goose biles and duck bile aresubstituted for the chicken gallbladder bile, with equivalent resultsbeing obtained.

EXAMPLE XIV 50 ml. of crude hog bile was lyophilized to yield a lightgreenish-yellow powder. This material was then dissolved in anhydrousmethanol, heated and filtered. Additional methanol was added and themethanol solution is then treated with sulfuric acid until completeesterification of the bile materials is obtained. The esterified bile isthen subjected to selective hydrolysis as by treatment with an alkalimetal base at an elevated temperature, in the presence of an alkalinebuffer whereby the pH of the reaction mixture is maintained at between10 and 11. The hydrolysis reaction is allowed to continue until the biteacid fraction of the bile is hydrolyzed. The resultant selectivityhydrolyzed bile is then transferred to a separatory funnel containing200 ml. of chloroform and 20 ml. of water and the funnel shaken for 1minute to permit phase separation. The lower chloroform phase isintensely greenish-yellow in color and contains substantially all thepigment and other impurities, while the upper alcoholic phase is a clearlight yellow in color and contains the bile acid fraction of the bile.The chloroform layer is drawn off and discarded.

Following the procedure of example V, but substituting the light yellowsolution obtained above for the solution of example ll, there isobtained the desired free bile acid.

EXAMPLE XV Following the procedure set forth in example II, butsubstituting equivalent amounts of hexane, dimethyl ether, acetone oracetic acid for methanol, equivalent results may be obtained.

EXAMPLE XV] Following the procedure set forth in example ll, butsubstituting equivalent amounts of toluene, methylene chloride, benzeneor methylene dichloride, for chloroform, equivalent results areobtained.

The invention may variously otherwise embodied within the scope of theappended claims.

I claim:

I. The method of producing substantially pure bile acids from naturalanimal bile, which comprises:

a. Substantially dehydrating the natural animal bile;

b. Esterifying the said dehydrated animal bile by treatment with asuitable esterification agent;

c. Subjecting the thus esterified animal bile to-a multiphasic organicsolvent system in one phase of which the bile acid fraction of saidanimal bile is soluble and the pigments and other impurities thereof arenot soluble, and in the remaining phases of which, the said pigments andother impurities are soluble and the bile acid fraction is not soluble;

d. Separating and recovering the bile acid fraction of the animal bile;

e. Treating the said bile acid fraction with a suitable Group Ila metalsalt to obtain the corresponding Group lla metal bile acid salts;

f. Treating said bile acid salts with an organic solvent in which saidbile acid salts possess differential sol ubility properties, to causethe separation of the desired bile acid salt;

g. Recovering said separated bile acid salt and subjecting said salt todissociation by treatment with a mineral acid; and

h. Recovering the desired free bile acid.

2. The method of producing substantially pure 3a,7a-dihydroxySB-cholanicacid from natural animal bile, which comprises:

a. Substantially dehydrating natural animal bile derived from chickens,turkeys, geese, pheasant, pidgeon or duck;

b. Esterifying said dehydrated bile by treatment with a suitable organicesterification agent;

c. Subjecting the thus esterified bile to a multiphasic organic solventsystem containing at least two phases, in one phase of which the bileacid fraction of said natural animal bile is soluble and the pigmentsand other impurities thereof are not soluble, and in the remainingphases of which the said bile acids are not soluble and the pigments andother impurities are soluble;

d. Separating and recovering the bile acid fraction of said animal bile;

e. Treating the thus recovered bile acids with a Group [In metal salt,said Group Ila metal selected from the group consisting of calcium,barium and strontium, to yield the corresponding Group [la metal bileacid salts;

f. Treating said Group [la metal acid salts with an organic solvent inwhich the Group metal salt of 3a,7a-dihydroxy-SB-cholanic acid is notsoluble, and in which the remaining group [I0 metal bile acid salts aresoluble;

g. Recovering the Group [Ia metal salt of 3a,7a-dihydroxy-SB-cholanicacid and subjecting it to dissociation by treatment with a mineral acid;and

h. Recovering the resultant free 3a,7a-dihydroxy-5B- cholanic acid.

3. The method of claim 1, wherein in step c. the multiphasic organicsolvent system is one selected from the group consisting of halogenatedhydrocarbon: alcohol; aromatic organic solvent: alcohol; aromaticorganic solvent: alkane; ether: alcohol; and halogenated hydrocarbon:ketone.

4. The method of claim I, wherein in step f., the organic solvent isselected from the group consisting of an alcohol, ethyl acetate, hexaneand acetic acid.

5. The method of claim 2, wherein in step c., the multiphasic organicsolvent system is selected from the group consisting of halogenatedhydrocarbon: alcohol; aromatic organic solvent: alcohol; aromaticorganic solvent: alkane; ether: alcohol; and halogenated hydrocarbon:ketone.

6. The method of claim 2, wherein in step f. the organic solvent isselected from the group consisting of an alcohol, ethyl acetate, hexaneand acetic acid.

7. The method of producing substantially pure bile acids from naturalanimal bile, which comprises:

a. Substantially dehydrating the natural animal bile;

b. Esterifying the said dehydrated animal bile;

c. Treating the thus esterified animal bile with a multiphasic organicsolvent system, in one phase of which the bile acid fraction of saidanimal bile is soluble and the pigments and other impurities of saidanimal bile are not soluble, and in the remaining phases of said solventsystem, the said pigments and impurities are soluble and the said bileacid fraction is not soluble;

d. Removing the pigment and other impurity fraction of the animal bile;

e. Treating the remaining bile acid fraction with a suitable salt toobtain the corresponding bile acid salts, which possess differentialsolubilities in an organic solvent; Treating said bile acid salts withan organic solvent, in which said bile acid salts possess differentialsolubility properties to cause separation of the desired bile acid salt;

g. Recovering the said separated bile acid salt;

h. Dissociating said separated bile acid salt to yield the desired freebile acid.

8. The method of claim 7 wherein in step e. the suitable salt is a Grouplla metal salt.

9. The Group Ila metal salts of the bile acids produced in accordancewith the method of claim 1, steps a through c.

10. The method of producing substantially pure3a,7a-dihydroxy-SB-cholanic acid from natural animal bile, whichcomprises a. Substantially dehydrating natural animal bile derived frombirds;

b. Esterifying said dehydrated bile by treatment with a suitable organicesterification agent;

c. Subjecting the thus esterified bile to a multiphasic organic solventsystem selected from the group consisting of halogenated hydrocarbon:alcohol; aromatic organic solvent: alcohol; aromatic organic solvent:alkane, ether: alcohol; and halogenated hydrocarbon: ketone; to separatethe pigment and other impurity fraction of said bile from the bile acidfraction thereof;

d. Treating the said bile acid fraction to obtain the corresponding freebile acids;

e. Treating said free bile acids with a Group Ila metal salt to obtainthe corresponding Group lla metal bile acid salts;

Treating said Group Ila metal bile acid salts with an organic solventselected from the group consisting of alcohols, ethyl acetate, hexaneand acetic acid to cause separation of the desired Group Ila metal saltof SaJa-dihydroxy-SB-cholanic acid and treating same to yield thedesired, substantially pure 3a.7a-dihydroxy-5B-cholanic acid.

H. The method of claim 1, wherein in step (e). the Group lla metal saltis selected from the group consisting of barium. calcium and strontium.

12. The method of claim I wherein in step (e). the Group lla metal saltis a barium salt.

13. The method of claim 1 wherein in step (e), the Group lla metal saltis a barium halide salt.

14. The method of claim 2 wherein in step (e). the Group lla metal saltis a barium halide salt.

15. The method of claim 7 wherein in step (e), the natural animal bileis derived from birds.

l6. The Group lla metal salts of the bile acids produced with the methodof claim 2, steps (a) through (e).

17. The bile acid salts obtained in accordance with the method of claim7, steps (a) through (e).

18. The method of producing substantially pure bile acids from naturalbile. which comprises:

a. Substantially dehydrating said natural animal bile;

b. Esterifying said substantially dehydrated natural animal bile bytreatment with a suitable organic esterification agent;

c. Suhjeeting said esterified natural animal bile to a multiphasicorganic solvent system in one phase of which the bile acid fraction ofsaid animal bile is soluble and the pigments and other impuritiesthereof are not soluble, and in the remaining phases of which the saidpigments and other impurities are soluble and the bile acid fraction isnot soluble;

d. Separating and recovering the substantially pigment and impurity-freebile acid fraction of the natural animal bile; and

e. Treating said bile acid fraction to isolate and recover the desired,substantially pure, individual bile acid components of said bile acidfraction.

t l i t

1. THE METHOD OF PRODUCING SUBSTANTIALLY PURE BILE ACIDS FROM NATURALANIMAL BILE, WHICH COMPRISES A. SUBSTANTIALLY DEGYDRAWTING THE NATURALANIMAL BILE, B. ESTERFIYING THE SAID DEHYDRATED ANIMAL BILE TREATMENTWITH A SUITABLE ESTERIFICATION AGENT C. SUBJECTING THE THUS ESTERFIEDANIMAL BILE TO A MULTIPHASIC ORGANIC SOLVENT SYSTEM IN ONE PHASE OFWHICH THE BILE ACID FRACTION OF SAID ANIMAL BILE IS SOLUBLE AND THEPIGMENTS AND THE OTHER IMPUTITIES THEREOF ARE NOT SOLUBLE, AND IN THEREMAINING PHASE OF WHICH, THE SAID PIGMENTS AND OTHER IMPURITIES ARESOLUBLE AND THE BILE ACID FRACTION IS NOT SOLUBLE, D. SEPARATING ANDRECOVERING THE BILE ACID FRACTION OF THE ANIMAL BILE, E. TREATING THESAID BILE ACID FRACTION WITH A SUITABLE GROUP LLA METAL SALT TO OBTAINTHE CORRESPONDING GROUP LLA METAL BILE ACID SALTS, F. TREATING SAID BILEACID WITH AN ORGANIC SOLVENT IN WHICH SAIDBILE POSSESS DIFFERENTIALSOLUBILITY PROPERITIES, TO CAUSE THE SEPARATION OF THE DESIRED BILE ACIDSALT, G. RECOVERING SAID SEPARATED BILE ACID SALT AND SUBJECTING SAIDSALT TO DISSOCATION BY TREATMENT WITH A MINERAL ACID, AND H. RECOVERINGTHE DESIRED FREE BILE ACID.
 2. The method of proDucing substantiallypure 3 Alpha ,7 Alpha -dihydroxy5 Beta -cholanic acid from naturalanimal bile, which comprises: a. Substantially dehydrating naturalanimal bile derived from chickens, turkeys, geese, pheasant, pidgeon orduck; b. Esterifying said dehydrated bile by treatment with a suitableorganic esterification agent; c. Subjecting the thus esterified bile toa multiphasic organic solvent system containing at least two phases, inone phase of which the bile acid fraction of said natural animal bile issoluble and the pigments and other impurities thereof are not soluble,and in the remaining phases of which the said bile acids are not solubleand the pigments and other impurities are soluble; d. Separating andrecovering the bile acid fraction of said animal bile; e. Treating thethus recovered bile acids with a Group IIa metal salt, said Group IIametal selected from the group consisting of calcium, barium andstrontium, to yield the corresponding Group IIa metal bile acid salts;f. Treating said Group IIa metal acid salts with an organic solvent inwhich the Group IIa metal salt of 3 Alpha ,7 Alpha -dihydroxy-5 Beta-cholanic acid is not soluble, and in which the remaining group IIametal bile acid salts are soluble; g. Recovering the Group IIa metalsalt of 3 Alpha ,7 Alpha -dihydroxy-5 Beta -cholanic acid and subjectingit to dissociation by treatment with a mineral acid; and h. Recoveringthe resultant free 3 Alpha ,7 Alpha -dihydroxy-5 Beta -cholanic acid. 3.The method of claim 1, wherein in step c. the multiphasic organicsolvent system is one selected from the group consisting of halogenatedhydrocarbon: alcohol; aromatic organic solvent: alcohol; aromaticorganic solvent: alkane; ether: alcohol; and halogenated hydrocarbon:ketone.
 4. The method of claim 1, wherein in step f., the organicsolvent is selected from the group consisting of an alcohol, ethylacetate, hexane and acetic acid.
 5. The method of claim 2, wherein instep c., the multiphasic organic solvent system is selected from thegroup consisting of halogenated hydrocarbon: alcohol; aromatic organicsolvent: alcohol; aromatic organic solvent: alkane; ether: alcohol; andhalogenated hydrocarbon: ketone.
 6. The method of claim 2, wherein instep f. the organic solvent is selected from the group consisting of analcohol, ethyl acetate, hexane and acetic acid.
 7. The method ofproducing substantially pure bile acids from natural animal bile, whichcomprises: a. Substantially dehydrating the natural animal bile; b.Esterifying the said dehydrated animal bile; c. Treating the thusesterified animal bile with a multiphasic organic solvent system, in onephase of which the bile acid fraction of said animal bile is soluble andthe pigments and other impurities of said animal bile are not soluble,and in the remaining phases of said solvent system, the said pigmentsand impurities are soluble and the said bile acid fraction is notsoluble; d. Removing the pigment and other impurity fraction of theanimal bile; e. Treating the remaining bile acid fraction with asuitable salt to obtain the corresponding bile acid salts, which possessdifferential solubilities in an organic solvent; f. Treating said bileacid salts with an organic solvent, in which said bile acid saltspossess differential solubility properties to cause separation of thedesired bile acid salt; g. Recovering the said separated bile acid salt;h. Dissociating said separated bile acid salt to yield the desired freebile acid.
 8. The method of claim 7 wherein in step e. the suitable saltis a Group IIa metal salt.
 9. The Group IIa metal salts of the bileacids produced in accordance with the method of claim 1, steps a throughe.
 10. The method of producing substantially pure 3 Alpha ,7 Alpha-dihydroxy-5 Beta -cholanic acid from natural animal bile, whichcomprises a. Substantially dehydrating natural animal bile derived frombirds; b. Esterifying said dehydrated bile by treatment with a suitableorganic esterification agent; c. Subjecting the thus esterified bile toa multiphasic organic solvent system selected from the group consistingof halogenated hydrocarbon: alcohol; aromatic organic solvent: alcohol;aromatic organic solvent: alkane, ether: alcohol; and halogenatedhydrocarbon: ketone; to separate the pigment and other impurity fractionof said bile from the bile acid fraction thereof; d. Treating the saidbile acid fraction to obtain the corresponding free bile acids; e.Treating said free bile acids with a Group IIa metal salt to obtain thecorresponding Group IIa metal bile acid salts; f. Treating said GroupIIa metal bile acid salts with an organic solvent selected from thegroup consisting of alcohols, ethyl acetate, hexane and acetic acid tocause separation of the desired Group IIa metal salt of 3 Alpha ,7 Alpha-dihydroxy-5 Beta -cholanic acid and treating same to yield the desired,substantially pure 3 Alpha ,7 Alpha -dihydroxy-5 Beta -cholanic acid.11. The method of claim 1, wherein in step (e), the Group IIa metal saltis selected from the group consisting of barium, calcium and strontium.12. The method of claim 1 wherein in step (e), the Group IIa metal saltis a barium salt.
 13. The method of claim 1 wherein in step (e), theGroup IIa metal salt is a barium halide salt.
 14. The method of claim 2wherein in step (e), the Group IIa metal salt is a barium halide salt.15. The method of claim 7 wherein in step (e), the natural animal bileis derived from birds.
 16. The Group IIa metal salts of the bile acidsproduced with the method of claim 2, steps (a) through (e).
 17. Thebiled acid salts obtained in accordance with the method of claim 7,steps (a) through (e).
 18. The method of producing substantially purebile acids from natural bile, which comprises: a. Substantiallydehydrating said natural animal bile; b. Esterifying said substantiallydehydrated natural animal bile by treatment with a suitable organicesterification agent; c. Subjecting said esterified natural animal bileto a multiphasic organic solvent system in one phase of which the bileacid fraction of said animal bile is soluble and the pigments and otherimpurities thereof are not soluble, and in the remaining phases of whichthe said pigments and other impurities are soluble and the bile acidfraction is not soluble; d. Separating and recovering the substantiallypigment and impurity-free bile acid fraction of the natural animal bile;and e. Treating said bile acid fraction to isolate and recover thedesired, substantially pure, individual bile acid components of saidbile acid fraction.